Cytokines function as protein mediators that play a critical role in host defense and serve as a communication link between cells of native and acquired immunity. Cells respond to cytokines with specific biological changes that are dependent on the activation of new gene expression.
Studies of the mechanism by which signals are signals are mediated from the receptor to the nucleus by the interferon cytokines have revealed the activation of latent cytoplasmic transcription factors that subsequently translocate to the nucleus (Bromberg, BioEssays, 2001, 23, 161-169).
The proteins of the STAT family (signal transducers and activators of transcription) are latent transcription factors that are abundantly expressed in many cell types. STATs are activated by phosphorylation on a single tyrosine in response to extracellular ligands. An active STAT dimer is formed through reciprocal interactions between the SH2 domain of one monomer and the phosphorylated tyrosine of the other monomer. The dimers accumulate in the nucleus, recognize specific DNA elements in the promoters of genes and activate transcription so that growth control and survival of normal cells in a developing or adult mammal are carefully balanced. Many of the signals that influence this balance are delivered by circulating polypeptides, whose binding to cell surface receptors governs gene-specific transcription. It has been shown that human cancer cells have lost control of these signaling mechanisms. In addition to persistent unregulated mitogenic signaling, the lack of suppressive signals is also critical in the development of cancers (Bromberg, BioEssays, 2001, 23, 161-169).
STAT 6 (also known as interleukin 4-STAT) was cloned and mapped to chromosome 12q13 (Leek et al., Cytogenet. Cell Genet., 1997, 79, 208-209; Quelle et al., Mol. Cell. Biol., 1995, 15, 3336-3343). Nucleic acid sequences encoding STAT 6 are disclosed and claimed in U.S. Pat. No. 5,710,266 (McKnight and Hou, 1998).
STAT 6 is primarily expressed as a 4 kb transcript in hematopoietic cells and expressed variably in other tissues (Quelle et al., Mol. Cell. Biol., 1995, 15, 3336-3343). A unique truncated isoform of STAT 6 is expressed in mast cells (Sherman, Immunol. Rev., 2001, 179, 48-56). Disclosed and claimed in PCT publication WO 99/10493 are nucleic acid sequences encoding variants of STAT 6 known as STAT 6b and STAT 6c as well as vectors comprising said nucleic acid sequences (Patel et al., 1999).
STAT 6 is an integral transcription factor involved in interleukin 4 and interleukin 13 signaling. Following activation of their respective receptors, interleukin 4 and interleukin 13 cause their common interleukin 4 receptor alpha chain to become phosphorylated by JAK3 and to subsequently bind to STAT 6. STAT 6 is then phosphorylated by JAK1, homodimerizes and translocates to the nucleus where it binds interleukin 4 response elements and initiates the transcription of a number of genes including IgE (Danahay et al., Inflamm. Res., 2000, 49, 692-699).
STAT 6 is involved in key pathological mechanisms in rheumatoid arthritis which operate in early and late stages of the disease (Muller-Ladner et al., J. Immunol., 2000, 164, 3894-3901).
Ghilardi et al. have found that STAT 6 interacts with an isoform of the leptin receptor (OB-R) and is thus, a potential mediator of the anti-obesity effects of leptin (Ghilardi et al., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 6231-6235).
STAT 6 knockout mice are viable and develop normally with the exception that interleukin 4 functions are eliminated (Ihle, Curr. Opin. Cell Biol., 2001, 13, 211-217). Additionally, STAT 6 knockout mice fail to develop antigen-induced airway hyper-reactivity in a model of airway inflammation (Kuperman et al., J. Exp. Med., 1998, 187, 939-948). Inhibition of STAT 6 is expected to attenuate the allergic response and thus, represents an attractive target for drug discovery strategies (Hill et al., Am. J. Respir. Cell Mol. Biol., 1999, 21, 728-737).
Small molecule inhibitors of STAT 6 are disclosed and claimed in PCT publication WO 00/27802 and Japanese Patent JP 2000229959 (Eyermann et al., 2000; Inoue et al., PCT, 2000, Abstract only). Disclosed and claimed in U.S. Pat. No. 6,207,391 are methods for screening modulators of STAT 6 binding to a STAT 6 receptor (Wu and McKinney, 2001).
Wang et al. have demonstrated targeted disruption of STAT 6 DNA-binding activity by a phosphorothioate cis-element decoy oligonucleotide (Wang et al., Blood, 2000, 95, 1249-1257).
Hill et al. have used a series of homologous human and murine antisense oligonucleotides targeting STAT 6 to interrupt interleukin 4 and interleukin 13 signaling and attenuate germline C-epsilon transcription in vitro (Hill et al., Am. J. Respir. Cell Mol. Biol., 1999, 21, 728-737). Subsequently, the in vitro and in vivo pharmacology of three of the antisense oligonucleotides used in the latter study was investigated. Although the oligonucleotides downregulated STAT 6 mRNA, their action was not sufficient to influence alterations in mRNA levels (Danahay et al., Inflamm. Res., 2000, 49, 692-699).
Currently, there are no known therapeutic agents that effectively inhibit the synthesis of STAT 6. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting STAT 6 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of STAT 6 expression.
The present invention provides compositions and methods for modulating STAT 6 expression.